Oral Contraceptive Steroids Promote Papillary Thyroid Cancer Metastasis by Targeting Angiogenesis and Epithelial-Mesenchymal Transition

Thyroid cancer is the most prevalent type of endocrine malignancy with the highest incidence rate among women under 45 years old. Ethinylestradiol (EE) and levonorgestrel (LNG) are two steroid components of low-dose oral contraceptives used all over the world. In this study, we aimed to examine the possible effects of the combination of these two steroids on metastasis and angiogenic factors in BCPAP papillary thyroid cancer (PTC) cell line. After treatment of BCPAP cells with the combination of 20 nM EE and 90 nM LNG, mRNA expression levels of long noncoding RNAs HOTAIR and MALAT1, angiogenic and antiangiogenic gene markers VEGFA and THBS1, and epithelial-mesenchymal transition (EMT) biomarkers CDH1, CDH2, FN1, and VIM were analyzed by real-time PCR. Additionally, the protein expression of VEGFA was semiquantified by Western blotting. Results showed that the combination of LNG and EE significantly elevated the level of VEGFA protein and mRNA expression of VEGFA, MALAT1, HOTAIR, CDH2, FN1, and VIM genes while decreased CDH1 gene expression but had no marked effect on the expression of THBS1 gene in comparison with the control group. Also, our results suggest that LNG and EE may increase the metastatic and migratory properties of BCPAP cells via modulating angiogenic and EMT biomarkers. These data may highlight the potential of exogenous steroids in the advancement of PTC tumors.

hyroid cancer (TC) is the most common type of endocrine malignancies and its incidence is growing worldwide. TC incidence is the highest among women aged under 45 years old (1). It has been revealed that TC is the second most incident cancer type in pregnant women after breast cancer (2). Papillary thyroid carcinoma (PTC) is the most common form of thyroid carcinoma comprising around 70% of all cases (3). PTC often develops at younger ages and has 2.9 fold higher incidence in women than men (4). Higher incidence of PTC in younger women could propose a role for sex hormones especially estrogen and progestins (whether endogenous or exogenous) in the development and progression of this type of cancer (5). Low dose (LD) contraceptives comprising levonorgestrel (LNG) and ethinylestradiol (EE) are widely used as birth control tablets in Iran and most European countries (6,7). Angiogenesis is a process by which tumor cells achieve their vast need for oxygen and nutrients supply. Vascular endothelial growth factor (VEGF) is the main protein responsible for the angiogenesis process, and its expression is elevated during this process in tumors. Trombospondin1 (THBS1) encodes a protein involved in reversing the above-mentioned process and is an anti-angiogenic factor (8). Long non-coding RNAs (lncRNAs) are more than 200 nucleotides long RNAs that have implications in the regular development and tumorigenesis process (9). Metastasis-associated lung adenocarcinoma transcript1 (MALAT1) encodes the lncRNA transcript that regulates the VEGF-mediated angiogenesis process (9). HOX transcript antisense RNA (HOTAIR) encodes lncRNA that regulates many cellular processes like proliferation, migration, and also angiogenesis process through regulating VEGF (10,11). Epithelial-mesenchymal transition (EMT) is a process with a critical role in the migration and metastasis of cancer cells (12).
Fibronectin1 (FN1), as a component of the extracellular matrix (ECM), plays a pivotal in the EMT process (13). Vimentin (VIM), as a component of the cytoskeleton, could be suggested as a biomarker for the EMT process (14), and cadherin 1 (CDH1) is a key protein involved in the EMT process due to its functions in cell-cell interactions, and reduced expression of CDH1 gene is associated with EMT initiation (15). Cadherin 2 (CDH2) is another calcium-dependent adhesion molecule that is elevated during EMT causing the interruption of cell-cell interactions (16). Our previous work demonstrated that LNG in combination with EE could induce proliferation and invasion of BCPAP cells while inhibited apoptosis of these cancer cells (1). In this study, we aimed to study the effects of combined LNG and EE (as LDcombined oral contraceptives, OCPs) on angiogenesis and EMT processes in PTC cell line, BCPAP.

Quantitative real-time polymerase chain reaction (QRTPCR)
To quantify the expression of our tested genes using QRTPCR method, BCPAP cells were seeded

Western blotting
For analyzing the expression of VEGFA protein, BCPAP cells were seeded in 6-well culture plates at a density of 25×10 4 cells/well. As mentioned before, the cells in the treatment group were exposed to 20 nM EE and 90 nM LNG, but the control cells were just treated with the normal medium for 48 h. Next, the treated cells were TGTCTTTCAGCAAGGACTGGT TGCTTACATGTCTCGATCCCAC   (21,22).

Statistical analysis
All data are expressed as mean ± SD, and were repeated at least three times. Statistical significance and differences between groups were analyzed using Student's t-test, and Mann-Whitney U test. P < 0.05 was considered a significant value. The data were analyzed using GraphPad Prism 7 software.

Effect of the combination of LNG and EE on angiogenesis-related factors expression
As depicted in Figures 1A and B

Effect of the combination of LNG and EE on EMT gene markers expression
The expression levels of four key genes involved in the process of EMT were further measured to evaluate the implication of this process in the metastatic properties of LNG and EE in BCPAP cells. Figure 3 shows the effect of the